Thermodynamic Origins of Sequence-Recognition in Ligand-DNA Interactions

配体-DNA 相互作用中序列识别的热力学起源

• 批准号: 9728720
• 负责人: David Graves
• 金额: $ 24万
• 依托单位: University of Mississippi
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728720&HistoricalAwards=false
• 关键词: Thermodynamic; Origins; Sequence; Recognition; Ligand

9728720 Graves The focus of this research is to gain insight into the mechanisms through which small molecules and proteins exert sequence-selective binding to nucleic acids. Efforts will be directed towards correlating the thermodynamic properties associated with these interactions with both DNA sequence and ligand structure. Recent studies have demonstrated the DNA binding enthalpies and entropies observed for actinomycin D-DNA interactions to be markedly influenced not only by the base-sequence at the intercalation site but also by the bases that flank the intercalation site. These observations will be extended to explore the effects of subtle changes in the DNA base sequences that flank the ligand binding site and their relationship to changes in the thermodynamic binding mechanism(s). Analyses of thermodynamic binding properties will be coupled to examination of the concomitant ligand-DNA structures resulting from interactions of ligands with specific DNA base sequences providing linkage between the thermodynamic mechanisms associated with complex formation and the structural geometry of the ligand-DNA complex. Various biophysical methods including absorption spectroscopy and isothermal microtitration calorimetry will be used to examine the interactions of actinomycin D with oligonucleotides that have been designed specifically to harbor a single centrally located actinomycin D binding site and varied neighboring and next-neighboring flanking base sequences. The binding site sequence and bases which flank the binding site will be systematically changed to determine their influence on the thermodynamic mechanism of complex formation. From these studies, insight into the thermodynamic origins of DNA sequence-recognition will be discerned. Structural details of the ligand-DNA complex will be examined by high-resolution nuclear magnetic resonance methods and correlated with the thermodynamic data to provide a novel paradigm for the "thermodynamic origins" of sequence-recognition. Mol ecular recognition is one of the central problems in understanding the mechanistic processes that govern a wide variety of biochemical and molecular biological events that take place in the cell. Hence, an understanding of the forces that govern the sequence specific interactions of small molecules and proteins with DNA is pivotal towards an overall comprehension of basic biochemical processes such as replication, transcription, recombination, and DNA repair. The focus of this research is to determine and characterize the chemical and/or structural determinants on both the target DNA and ligand responsible for sequence-selective interactions.

9728720 Graves本研究的重点是深入了解小分子和蛋白质与核酸序列选择性结合的机制。 努力将被导向与这些相互作用与DNA序列和配体结构相关的热力学性质。 最近的研究表明，放线菌素D-DNA相互作用中观察到的DNA结合势和熵不仅受到嵌入位点碱基序列的显著影响，而且还受到嵌入位点侧翼碱基的显著影响。 这些观察结果将被扩展，以探索配体结合位点侧翼的DNA碱基序列的细微变化的影响及其与热力学结合机制变化的关系。 热力学结合特性的分析将被耦合到检查伴随的配体-DNA结构产生的配体与特定的DNA碱基序列的相互作用，提供与复合物的形成和配体-DNA复合物的结构几何形状相关的热力学机制之间的联系。 各种生物物理方法，包括吸收光谱法和等温微量滴定量热法将被用来检查放线菌素D与寡核苷酸的相互作用，这些寡核苷酸被专门设计为具有一个单一的位于中心的放线菌素D结合位点和不同的相邻和下一个相邻的侧翼碱基序列。 结合位点序列和结合位点侧翼的碱基将被系统地改变，以确定它们对复合物形成的热力学机制的影响。 从这些研究中，深入了解DNA序列识别的热力学起源将被辨别出来。 配体-DNA复合物的结构细节将通过高分辨率核磁共振方法进行检查，并与热力学数据相关联，为序列识别的“热力学起源”提供新的范式。 分子识别是理解细胞中发生的各种生物化学和分子生物学事件的机制过程的中心问题之一。 因此，对控制小分子和蛋白质与DNA的序列特异性相互作用的力的理解对于全面理解基本的生物化学过程如复制、转录、重组和DNA修复至关重要。 本研究的重点是确定和表征负责序列选择性相互作用的靶DNA和配体上的化学和/或结构决定簇。